Synergistic investigation of natural and synthetic C1-trophic microorganisms to foster a circular carbon economy.

A true circular carbon economy must upgrade waste greenhouse gases. C1-based biomanufacturing is an attractive solution, in which one carbon (C1) molecules (e.g. CO2, formate, methanol, etc.) are converted by microbial cell factories into value-added goods (i.e. food, feed, and chemicals). To render C1-based biomanufacturing cost-competitive, we must adapt microbial metabolism to perform chemical conversions at high rates and yields. To this end, the biotechnology community has undertaken two (seemingly opposing) paths: optimizing natural C1-trophic microorganisms versus engineering synthetic C1-assimilation de novo in model microorganisms. Here, we pose how these approaches can instead create synergies for strengthening the competitiveness of C1-based biomanufacturing as a whole.

Economical production of Pichia pastoris single cell protein from methanol at industrial pilot scale.

BACKGROUND: Methanol, synthesized from CO2, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production. RESULTS: In the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine). CONCLUSIONS: This study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.

Assessment of prokaryotic communities in Southwestern Atlantic deep-sea sediments reveals prevalent methanol-oxidising Methylomirabilales.

Continental slopes can play a significant contribution to marine productivity and carbon cycling. These regions can harbour distinct geological features, such as salt diapirs and pockmarks, in which their depressions may serve as natural sediment traps where different compounds can accumulate. We investigated the prokaryotic communities in surface (0-2 cm) and subsurface (18-20 or 22-24 cm) sediments from a salt diapir and pockmark field in Santos Basin, Southwest Atlantic Ocean. Metabarcoding of 16 samples revealed that surface sediments were dominated by the archaeal class Nitrososphaeria, while the bacterial class Dehalococcoidia was the most prevalent in subsurface samples. Sediment strata were found to be a significant factor explaining 27% of the variability in community composition. However, no significant difference was observed among geomorphological features. We also performed a metagenomic analysis of three surface samples and analysed the highest quality metagenome-assembled genome retrieved, which belonged to the family CSP1-5, phylum Methylomirabilota. This non-methanotrophic methylotroph contains genes encoding for methanol oxidation and Calvin Cycle pathways, along with diverse functions that may contribute to its adaptation to deep-sea habitats and to oscillating environmental conditions. By integrating metabarcoding and metagenomic approaches, we reported that CSP1-5 is prevalent in the sediment samples from Santos Basin slope, indicating the potential importance of methanol metabolism in this region. Finally, using a phylogenetic approach integrating 16S rRNA sequences assigned to Methylomirabilota in this study with those from a public database, we argued that CSP1-5 public sequences might be misclassified as Methylomirabilaceae (the methanotrophic clade) and, therefore, the role of these organisms and the methanol cycling could also be neglected in other environments.

Production of codon-optimized Human papillomavirus type 52 L1 virus-like particles in Pichia pastoris BG10 expression system.

Cervical cancer caused by Human papillomavirus (HPV) is one of the most common causes of cancer death in women worldwide. Even though the disease can be avoided by immunization, the expensive price of HPV vaccines makes it hard to be accessed by women in middle-low-income countries. Thus, the development of generic HPV vaccines is needed to address inequalities in life-saving access. This study aimed to develop the HPV52 L1 VLP-based recombinant vaccine using Pichia pastoris expression system. The l1 gene was codon-optimized based on P. pastoris codon usage resulting CAI value of 0.804. The gene was inserted into the pD902 plasmid under the regulation of the AOX1 promoter. The linear plasmid was transformed into P. pastoris BG10 genome and screened in YPD medium containing zeocin antibiotic. Colony of transformant that grown on highest zeocin concentration was characterized by genomic PCR and sequencing. The positive clone was selected and expressed using BMGY/BMMY medium induced with various methanol concentrations. The SDS-PAGE and Western blot analyses showed that 55 kDa L1 protein was successfully expressed using an optimum concentration of 1% methanol. The self-assembly of HPV52 L1 protein was also proven using TEM analysis. Moreover, we also analyzed the B-cell epitope of HPV52 L1 protein based on several criteria, including antigenicity, surface accessibility, flexibility, and hydrophilicity. We assumed that epitope 476GLQARPKLKRPASSAPRTSTKKKKV500 could be developed as an epitope-based vaccine with a neutralizing antibody response toward HPV52 infection. Finally, our study provided the alternative for developing low-cost HPV vaccines, either VLP or epitope-based.

Biological isolation and characterization of Catharanthus roseus (L.) G. Don methanolic leaves extracts and their assessment for antimicrobial, cytotoxic, and apoptotic activities.

BACKGROUND: Biological synthesis of natural products from plants has made us an inspiring methodology in the field of science and biotechnology. METHODS: The methanolic extracts of Catharanthus roseus (L.) G. Don plant leaves (CrPLE) were extracted and characterized by utilizing the phytochemicals estimation, Thin-layer chromatography (TLC), and High-Performance Liquid Chromatography (HPLC) analysis; and further evaluation for an understanding of the biomedical uses of CrPLE was done. RESULTS: The evaluation of the seven phytochemicals designates the presence of secondary metabolites in the CrPLE. The CrPLE (test samples) exhibited the Catechin and Caffeic acid contents of 0.0055 and 0.0149 mg/g respectively. The CrPLE revealed the highest antimicrobial activity and showed a mortal effect against the tested microorganisms. Cytotoxicity of the breast cancer cell lines was exposed that CrPLE as a respectable anticancer specialist and metabolically vigorous cells. CONCLUSION: Consequently, the biological synthesized methanolic leaf extracts of the C. roseus plants would be appreciated and have incredible contributions to the field of medicinal applications.

Antifungal compounds, GC-MS analysis and toxicity assessment of methanolic extracts of Trichoderma species in an animal model.

Fungi of the genus Trichoderma have been marketed for the management of diseases of crops. However, some Trichoderma species may produce toxic secondary metabolites and it should receive due attention to ensure human safety. In this study, we investigated the in vitro antagonistic potential of T. asperellum AU131 and T. longibrachiatum AU158 as microbial biocontrol agents (MBCAs) against Fusarium xylarioides and the associated antagonistic mechanism with bioactive substances. Swiss albino mice were used to evaluate the in vivo toxicity and pathogenicity of T. asperellum AU131 and T. longibrachiatum AU158 methanolic extracts and spore suspensions, respectively, in a preliminary safety assessment for use as biofungicides. Gas Chromatography-Mass Spectrometry (GC-MS) was used to profile volatile organic metabolites (VOCs) present in the methanolic extracts. The agar diffusion assay of the methanolic extracts from both T. asperellum AU131 and T. longibrachiatum AU158 were effective at a concentration of 200 µg/mL (1×107 spores/mL), causing 62.5%, and 74.3% inhibition, respectively. A GC-MS analysis of methanolic extracts from both bioagents identified 23 VOCs which classified as alcohols, acids, sesquiterpenes, ketones and aromatic compounds. The oral administration of methanolic extracts and spore suspensions of each Trichoderma species to female Swiss albino mice over 14 days did not show any significant signs of toxicity, mortality or changes to body weight. It can be concluded that the tested spore suspensions and methanolic extracts were not pathogenic or toxic, respectively, when administered to Swiss albino mice at various doses.

Bio-conversion of methane into high profit margin compounds: an innovative, environmentally friendly and cost-effective platform for methane abatement.

Despite the environmental relevance of CH4 and forthcoming stricter regulations, the development of cost-efficient and environmentally friendly technologies for CH4 abatement is still limited. To date, one of the most promising solutions for the mitigation of this important GHG consists of the bioconversion of CH4 into bioproducts with a high profit margin. In this context, methanotrophs have been already proven as cell-factories of some of the most expensive products synthesized by microorganisms. In the case of ectoine (1000 $ kg-1), already described methanotrophic genera such as Methylomicrobium can accumulate up to 20% (ectoine wt-1) using methane as the only carbon source. Moreover, α-methanotrophs, such as Methylosynus and Methylocystis, are able to store bioplastic concentrations up to 50-60% of their total cell content. More than that, methanotrophs are one of the greatest potential producers of methanol and exopolysaccharides. Although this methanotrophic factory could be enhanced throughout metabolic engineering, the valorization of CH4 into valuable metabolites has been already consistently demonstrated under continuous and discontinuous mode, producing more than one compound in the same bioprocess, and using both, single strains and specific consortia. This review states the state-of-the-art of this innovative biotechnological platform by assessing its potential and current limitations.

Assessment of the endogenous respiration rate and the observed biomass yield for methanol-fed denitrifying bacteria under anoxic and aerobic conditions.

In this study, the endogenous respiration rate and the observed biomass yield of denitrifying methylotrophic biomass were estimated through measuring changes in denitrification rates (DNR) as a result of maintaining the biomass under methanol deprived conditions. For this purpose, activated sludge biomass from a full-scale wastewater treatment plant was kept in 10-L batch reactors for 8 days under fully aerobic and anoxic conditions at 20 °C without methanol addition. To investigate temperature effects, another biomass sample was placed under starvation conditions over a period of 10 days under aerobic conditions at 25 °C. A series of secondary batch tests were conducted to measure DNR and observed biomass yields. The decline in DNR over the starvation period was used as a surrogate to biomass decay rate in order to infer the endogenous respiration rates of the methylotrophs. The regression analysis on the declining DNR data shows 95% confidence intervals of 0.130 ± 0.017 day-1 for endogenous respiration rate under aerobic conditions at 20 °C, 0.102 ± 0.013 day-1 under anoxic conditions at 20 °C, and 0.214 ± 0.044 day-1 under aerobic conditions at 25 °C. Results indicated that the endogenous respiration rate of methylotrophs is 20% slower under anoxic conditions than under aerobic conditions, and there is a significant temperature dependency, with an Arrhenius coefficient of 1.10. The observed biomass yield value showed an increasing trend from approximately 0.2 to 0.6 when the starvation time increased from 0 to 10 days.

Assessment of active methanogenic archaea in a methanol-fed upflow anaerobic sludge blanket reactor.

Methanogenic archaea enrichment of a granular sludge was undertaken in an upflow anaerobic sludge blanket (UASB) reactor fed with methanol in order to enrich methylotrophic and hydrogenotrophic methanogenic populations. A microbial community assessment, in terms of microbial composition and activity-throughout the different stages of the feeding process with methanol and acetate-was performed using specific methanogenic activity (SMA) assays, quantitative real-time polymerase chain reaction (qPCR), and high-throughput sequencing of 16S ribosomal RNA (rRNA) genes from DNA and complementary DNA (cDNA). Distinct methanogenic enrichment was revealed by qPCR of mcrA gene in the methanol-fed community, being two orders of magnitude higher with respect to the initial inoculum, achieving a final mcrA/16S rRNA ratio of 0.25. High-throughput sequencing analysis revealed that the resulting methanogenic population was mainly composed by methylotrophic archaea (Methanomethylovorans and Methanolobus genus), being also highly active according to the RNA-based assessment. SMA confirmed that the methylotrophic pathway, with a direct conversion of methanol to CH4, was the main step of methanol degradation in the UASB. The biomass from the UASB, enriched in methanogenic archaea, may bear great potential as additional inoculum for bioreactors to carry out biogas production and other related processes.

Techno-economic assessment of integrating methanol or Fischer-Tropsch synthesis in a South African sugar mill.

This study considered an average-sized sugar mill in South Africa that crushes 300 wet tonnes per hour of cane, as a host for integrating methanol and Fischer-Tropsch synthesis, through gasification of a combined flow of sugarcane trash and bagasse. Initially, it was shown that the conversion of biomass to syngas is preferably done by catalytic allothermal gasification instead of catalytic autothermal gasification. Thereafter, conventional and advanced synthesis routes for both Methanol and Fischer-Tropsch products were simulated with Aspen Plus® software and compared by technical and economic feasibility. Advanced FT synthesis satisfied the overall energy demands, but was not economically viable for a private investment. Advanced methanol synthesis is also not viable for private investment since the internal rate of return was 21.1%, because it could not provide the steam that the sugar mill required. The conventional synthesis routes had less viability than the corresponding advanced synthesis routes.

[Inhibition of methanogenium by erythromycin and its domestation].

Erythromycin is a kind of antibiotic drugs with certain biological toxicity. In order to investigate the inhibitory effect of erythromycin on methanogens and its acclimation capacity, Anaerobic Toxicity Assay (ATA) and continuous experiment were conducted in anaerobic bottles and the Up-flow Anaerobic Sludge Blanket Reactor (UASB), respectively, to determine the accumulated methane production, ratio of methane production rate, COD removal efficiency, and methane content. The results showed that the methane production ratio was reduced to 56.1% in the presence of 150 mg x L(-1) of erythromycin and it was reduced by 99% when the erythromycin reached 250 mg x L(-1), indicating that the activity was completely inhibited. Keeping the erythromycin at an concentration of 20 mg x L(-1) in the process of continuous operation for 60d, the COD removal efficiency and methane content reached up to 81.4% and 64.2%, respectively. The results suggested that erythromycin had an inhibitory effect on methane bacteria, and the half inhibitory concentration was 150 mg x L(-1) (IC50:150 mg x L(-1)). The COD removal efficiency and methane content were increased by 15.13% and 22.05%, respectively, after domestication for 60 d.

Life cycle assessment of biofuel production from brown seaweed in Nordic conditions.

The use of algae for biofuel production is expected to play an important role in securing energy supply in the next decades. A consequential life cycle assessment (LCA) and an energy analysis of seaweed-based biofuel production were carried out in Nordic conditions to document and improve the sustainability of the process. Two scenarios were analyzed for the brown seaweed (Laminaria digitata), namely, biogas production (scenario 1) and bioethanol+biogas production (scenario 2). Potential environmental impact categories under investigation were Global Warming, Acidification and Terrestrial Eutrophication. The production of seaweed was identified to be the most energy intensive step. Scenario 1 showed better performance compared to scenario 2 for all impact categories, partly because of the energy intensive bioethanol separation process and the consequently lower overall efficiency of the system. For improved environmental performance, focus should be on optimization of seaweed production, bioethanol distillation, and management of digestate on land.

Factors affecting the methanol content and yield of plum brandy.

UNLABELLED: This study was conducted to determine the influence of plum cultivar, harvest year, and plum component on the methanol content and the yield of plum brandy. Seven plum cultivars (Geneva Mirabelle, French Damson, Pozegaca, Oblinaya, Early Golden, Lohr, and Rosy Gage) grown in the Finger Lakes fruit region of New York State were processed into mash and juice. The samples of plum mash or juice were fermented with commercial Red Star wine yeast Montrachet (Sachharomyces cerevisiae Davis 522) for 12 d. The fermented samples were distilled, and the distillates were analyzed for methanol, ethanol, and higher alcohols by high-performance liquid chromatography. Duncan's multiple range tests show significant differences in the methanol content and the yield of plum brandy made from 7 plum cultivars. The harvest year also had a significant effect on the methanol content and the yield of plum brandy. Student's t-test results indicate that plum juices gave a lower methanol content of brandy than plum mashes without significantly reducing the brandy yield. The results of the current research can be used by the industry to select the better plum cultivar and to adopt the process to improve the product yield and quality. PRACTICAL APPLICATION: The brandy industry can apply the results of the current research to improve product yield and to reduce the methanol content of plum brandy. The economic benefits to the brandy producers adopting the brandy production process will be significant due to the sales of new products with an acceptable level of methanol.

Dynamic flux balance analysis for pharmaceutical protein production by Pichia pastoris: human growth hormone.

The influence of methanol feeding rate on intracellular reaction network of recombinant human growth hormone (rhGH) producing Pichia pastoris was investigated at three different specific growth rates, namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04 h(-1) (MS-0.04) where Period-I (33 ≤ t <42 h) includes the early exponential growth phase; Period-II (42 ≤ t<48 h) is the exponential growth phase where the specific cell growth rate decreases; Period-III (48 ≤ t ≤51 h) is the exponential growth phase where rhGH concentration was the highest; and Period-IV (t>51 h) is the diminution phase for rhGH and cell synthesis. In Period-I, almost all of the formaldehyde entered the assimilatory pathway, at MS-0.02 and MS-0.03, whereas, at MS-0.04 high methanol feeding rate resulted in an adaptation problem. In Period-III, only at MS-0.02 co-carbon source sorbitol uptake-flux was active showing that sorbitol uptake does not affected from the predetermined feeding rate of methanol at µ(0)>0.02 h(-1). The biomass synthesis flux value was the highest in Period-I, -II and -III, respectively at MS-0.03 & MS-0.04, MS-0.04 and MS-0.02; whereas, rhGH flux was the highest in Period-I, -II, and -III, respectively at MS-0.03, MS-0.02 and MS-0.03. Based on the fluxes, Period-I should start with MS-0.03 methanol feeding rate and starting from the middle of Period-II methanol feeding rate should be shifted to MS-0.02.

The assessment of different operating strategies for minimising activated sludge deflocculation under temperature transient conditions.

Three operating strategies were tested for decreasing activated sludge deflocculation due to temperature shifts from 30 degrees to 45 degrees C: magnesium sludge enrichment, increased sludge retention time (33 d), and spikes of an easily degradable substrate (methanol). The temperature shifts were conducted sequentially in 4 parallel lab-scale sequencing batch reactors (SBRs) treating kraft pulp mill effluent. Three SBRs operated at an SRT = 20 days, and in one of them the sludge was not manipulated, thus, serving as a reference SBR. The temperature shift was associated with decreased soluble chemical oxygen demand (SCOD) removals, decreased sludge settleability and substrate removal capacity, and increased effluent suspended solids (ESS) and turbidity levels. The shift also increased the sludge specific respiration rates and reduced the sludge substrate removal capacity. Sludge deflocculation was assessed as floc solubilisation (increased effluent SCOD levels) and floc fragmentation (increase in effluent solids smaller than 50 microm). Mg enrichment of the sludge and methanol spikes reduced the ESS levels (in 9 and 25%), and the three operating strategies decreased effluent turbidity (in 22-35%) compared to the maximum levels from the non-manipulated reactor (44 mg ESS/L). The stronger sludge floc structure achieved by magnesium enrichment and a high sludge age of 33 days was unsuccessful in significantly decreasing deflocculation. The mechanisms involved in sludge deflocculation require further fundamental research.

Assessment of compatible solutes to overcome salinity stress in thermophilic (55 degrees C) methanol-fed sulfate reducing granular sludges.

High NaCl concentrations (25 g x L(-1)) considerably decreased the methanol depletion rates for sludges harvested from two lab-scale sulfate reducing UASB reactors. In addition, 25 gNaCl x L(-1) strongly affected the fate of methanol degradation, with clear increase in the acetate production at the expense of sulfide and methane production. The addition of different osmoprotectants, viz. glutamate, betaine, ectoine, choline, a mixture of compatible solutes and K+ and Mg2+, slightly increased methanol depletion rates for UASB reactors sludges. However, the acceleration in the methanol uptake rate favored the homoacetogenic bacteria, as the methanol breakdown was steered to the formation of acetate without increasing sulfate reduction and methane production rates. Thus, the compatible solutes used in this work were not effective as osmoprotectants to alleviate the acute NaCl toxicity on sulfate reducing granular sludges developed in methanol degrading thermophilic (55 degrees C) UASB reactors.

The effect of lithium chloride on the biooxidation of aqueous methanol/acetone mixtures.

Lithium chloride, more specifically the lithium cation, has been implicated in interference in biological systems. In the case of Escherichia coli, interference involves the Na+(Li+)/H+ antiporter transport system. The study reported here concerns the effects of LiCl on a mixed enrichment culture that is able to biodegrade both methanol and acetone under aerobic conditions. The results obtained using unsteady state continuous flow culture techniques demonstrate a significant disruptive effect of LiCl on culture performance. In addition, a reduction in the substrate-based biomass yield coefficient, which is a clear advantage as far as biotreatment process performance is concerned, also occurs. The ultimate fate of the LiCl was not determined.

Essential dynamics of reversible peptide folding: memory-free conformational dynamics governed by internal hydrogen bonds.

A principal component analysis has been applied on equilibrium simulations of a beta-heptapeptide that shows reversible folding in a methanol solution. The analysis shows that the configurational space contains only three dense sub-states. These states of relatively low free energy correspond to the "native" left-handed helix, a partly helical intermediate, and a hairpin-like structure. The collection of unfolded conformations form a relatively diffuse cloud with little substructure. Internal hydrogen-bonding energies were found to correlate well with the degree of folding. The native helical structure folds from the N terminus; the transition from the major folding intermediate to the native helical structure involves the formation of the two most C-terminal backbone hydrogen bonds. A four-state Markov model was found to describe transition frequencies between the conformational states within error limits, indicating that memory-effects are negligible beyond the nanosecond time-scale. The dominant native state fluctuations were found to be very similar to unfolding motions, suggesting that unfolding pathways can be inferred from fluctuations in the native state. The low-dimensional essential subspace, describing 69% of the collective atomic fluctuations, was found to converge at time-scales of the order of one nanosecond at all temperatures investigated, whereas folding/unfolding takes place at significantly longer time-scales, even above the melting temperature.

Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris.

Cost effective 13C/15N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The 13C/15N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut+) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (MutS) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for 13C-isotope labeling can be substantially reduced using the Mut+ strain compared to the MutS strain, as no 13C3-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for 15N-labeling by an optimal supply of 10 g/L (15NH4)2SO4 every 24 h.

Gas chromatographic assessment of alcoholyzed fatty acids from yeasts: a new chemotaxonomic method.

An alternative chemotaxonomic method to methanolysis was developed for gas chromatographic assessment of fatty acids in whole yeast cells. Clinical and reference strains of the medically important yeasts Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae were cultured for 48 h at 26 degrees C. Cellular lysis and transesterification were then performed with ethanol, propanol, butanol, or methanol. The relative recovery rates for cellular fatty acids, including the volatile acids C10:0 and C12:0, were similar after alcoholysis with ethanol, propanol, or butanol, while methanolysis gave lower recoveries of volatile fatty acids. Thus, after ethanolysis, the recovery of C10:0 acid (0.1, 1, and 10%) from a defined matrix (lyophilized Actinobacillus actinomycetemcomitans cells) varied from 97 to 102%, while the recovery of C10:0 after methanolysis varied from 49 to 75%. This indicated that with the frequently used methanolysis technique, there is a considerable loss of volatile fatty acids. These acids may be used as marker molecules for taxonomic differentiation between yeasts.